A number of laboratories, including those involved in this Program Project, have demonstrated that Adeno-associated virus (AAV) hold significant promise for the treatment of human disease. However, if the development of AAV as a vector is to proceed further, several issues must be addressed. Among them are the methods currently used to produce recombinant AAV virus, which are cumbersme and not amenable to large volume production. Previous attempts to develop packaging lines or adenovirus (Ad) vectors carrying AAV genes have floundered because there are large gaps in our understanding of the interaction of the AAV Rep proteins with cellular proteins, and in our understanding of how AAV gene expression is controlled duringproductive infections. For example, no information is available about the specific elements within the p19 and p40 AAV promoters that are important for transcription of for Rep mediated transactivation. To remedy this, we have recently begun studying the mechanism by which AAV controls its own gene expressionduring productive infections. We have found that AAV gene regulationis complex. At least one control loop has been uncovered that apparently is designed to maintain constant AAV p5 and p19 mRNA levles, presumably to maximize virus production. In addition, we have demonstrated that the p5 Rep proteins when bound to the p5 Rep binding element (RBE) can act both as repressors and transactivators of AAB transcription, depending on the target promoter. This kind of information is likely to be essential in the design of better production methods and therefore, this proposal willfocus on the molecular biology and biochemistry of AAV transcription. Specifically, we will: 1) Identify and characterize the basal and Rep responsive elements within the p19 and p40 promoters that control AAV transcrition, 2) Characterize the Re--SP1 and Rep 78-Rep 52 protein interactions, and 3) pruify and clone the cellular AAV activating protein (cAAP).